Randomized control trial to investigate compliance with, and impacts of, cow comfort recommendations on smallholder dairy farms in Kenya.

Smallholder dairy farmers (SDF) in developing countries often have limited understanding on the importance of cow comfort. We conducted a randomized controlled trial with 124 cows on 114 Kenyan SDF to determine the status of cow comfort, to assess compliance to farm-specific cow comfort recommendations, and to evaluate the impacts of the farm-specific interventions on cow comfort. On the first farm visit, stall dimensions and characteristics (e.g. stall base hardness and hygiene) were measured and categorized as adequate, marginal or inadequate/absent based on cow size. Where measurements were not adequate, farm-specific cow comfort recommendations were provided in written and oral form to the randomly allocated intervention group of farms (n = 74). On the second farm visit two months later, the same measurements were taken, and percent compliance to the recommendations was evaluated. A discomfort index was arithmetically calculated based on the stall base hardness (scale was 1-3 for soft to hard) and hygiene (scale was 1-5 for clean to dirty). Multivariable linear regression models were used to determine specific associations with the discomfort index. On the first visit, the mean stall base hardness and stall hygiene scores were 1.7 and 2.3, respectively, for a mean discomfort index of 4.0. Intervention farmers were given 3.9 comfort recommendations, on average, and complied with 2.1 recommendations, significantly improving the discomfort index at visit two to 3.3. The overall compliance to the recommendations was 49.0%. In a final model, the interaction between intervention group and visit number was significantly associated with discomfort index, indicating that after adjusting for baseline discomfort indices, the intervention led to better cow comfort. Specifically, bedding type and neck rail positioning were significantly associated with discomfort index. We concluded that farmers can substantially improve cow comfort on SDF by providing recommendations to them. Farm advisors should include cow comfort recommendations to SDF.

Evaluation of environmental and comfort improvements on affective welfare in heifer calves on smallholder dairy farms.

A controlled trial on zero-grazed smallholder dairy farms was conducted to determine the effect of environmental and comfort improvements on sucking and lying behaviours in heifer calves on Kenyan smallholder dairy farms. The study involved 187 heifer calves from 150 farms in two Kenyan counties, 75 farms per county. Farms in one county received animal welfare training and improvements in the calf pen that included: 1) placement of rubber mats on the lying area; 2) fixing gaps/holes in the flooring and roofing; and 3) attaching a rubber nipple on the wall of the calf pen. During the 16-month data collection period, bimonthly farm visits were used to collect data on lying time (using accelerometers) and other animal- and farm-level factors. Multilevel mixed-effects linear regression was used to model daily lying times and frequency of lying bouts, with the animal as a random effect. Over the visits, daily lying times and lying bout durations averaged 12.6-86.7 min/bout, respectively, while the median for the frequency of lying bouts was between 30-46/day. Provision of rubber nipples for non-nutritive sucking lowered proportions of cross-sucking, self-sucking and object-sucking behaviours slightly but not significantly. In a final daily lying time model, superficial lymph node enlargement, body condition score and use of wood shaving/ sawdust/ crop waste as beddings had positive associations. In contrast, group housing and rubber mat use had negative associations with daily lying time. In an interaction term, lying time was significantly higher for calves on clean versus dirty floors if the age was <190 days but this difference diminished significantly in older animals. In a second interaction term, lying time was lower for calves with leaking versus non-leaking roofs, regardless of the pen floor level, but lying time was higher on elevated than non-elevated floors if the roof was intact. In the final model of the frequency of lying bouts, the use of a rubber mat, the years of experience in dairy farming, and calf body weight had negative associations. In contrast, body condition score had a positive association. In an interaction, the frequency of daily lying bouts was lower on clean floors than dirty floors, irrespective of tethering status, but when the floor was dirty, the lying bouts were higher for animals not tethered than the ones sometimes tethered. We conclude that the comfort improvements enhanced the welfare and lying experience of heifer calves on smallholder dairy farms.

Relationships between type of hoof lesion and behavioral signs of lameness in Holstein cows housed in Canadian tiestall facilities.

Although foot pain can affect gait, the presence of a hoof lesion may or may not cause the cow to show visible changes in their gait. This can be dependent on the type and severity of the lesion; for example, the presence of a sole ulcer (SU) has been associated with increased gait scores, whereas digital dermatitis (DD) and sole hemorrhage (SH) have not. In tiestall facilities, gait scoring can be difficult to perform. An alternative method, known as stall lameness scoring (SLS), allows observers to assess cattle for lameness while they remain in their stall. Lameness is determined based on behavioral changes in weight bearing and foot positioning, which include: shifting weight, resting a foot, standing on the edge of the stall, and uneven weight bearing when stepping side to side. The aim of this study was to examine relationships between hoof lesions and these behavioral indicators. A total of 557 observations of SLS and corresponding hoof trimming records, collected during routine trimming events on 7 tiestall herds, were obtained. Trimming was performed by 2 trained hoof trimmers with good agreement on lesion identification, based on quizzes taken at the beginning and mid-way through the study. To ensure trimming had no effect on the behavioral indicators observed, SLS was always performed by a trained observer before trimming. Behavioral indicators focused on the hind limbs only; therefore, the analysis was confined to hind limb lesions using logistic regression to detect the presence of hoof lesion based on observations made during SLS. Seventy-five percent of observed cows had no SLS behavioral indicators, whereas, 11, 12, and 1% had 1, 2, and 3 behavioral indicators, respectively. At least one hind limb lesion was noted during trimming in 19% of cows, with the most common lesions being DD (7%), SU (6%), and SH (4%). A cow that was observed resting one foot and bearing weight unevenly when moving side to side had higher odds of having a hind limb hoof lesion than a cow not displaying these behaviors. When looking at specific hoof lesions, a cow observed resting one limb and bearing weight unevenly had higher odds of having a SU compared with those not displaying these behaviors. A cow observed shifting their weight from one foot to another had higher odds of having SH, and a cow observed bearing weight unevenly had higher odds of DD. Behavioral indicators in weight bearing and foot positioning can help identify cows in tiestalls with hind limb hoof lesions. Producers could routinely observe their cattle for these indicators to assist in the identification of cows that may require treatment. This could help reduce the duration of clinical lameness through earlier intervention.

Management factors associated with time-to-onset and fecal egg/oocyst counts of gastrointestinal parasites in heifer calves on Kenyan smallholder farms.

A prospective cohort study on zero-grazed smallholder dairy farms was conducted to determine factors associated with onset and counts of gastrointestinal parasitism in heifer calves. The researchers recruited 187 newborn heifer calves from 150 farms in Kenya. Over 16 months, farm visits every two months were used to collect rectal fecal samples and animal- and farm-level measures. Fecal samples underwent centrifugal fecal flotation with Sheather's sugar to determine counts of strongyle-type eggs and coccidia oocysts. Cox proportional hazard (Cox PH) analysis and mixed-effects negative binomial (MeNB) regression determined factors associated with time-to-onset and counts of strongyle-type eggs and coccidia oocysts, respectively (P < 0.05). The incidence rate of strongyles was 0.0011 cases/animal-day while coccidia was 0.0073 cases /animal-day. Incidence risks of strongyles and coccidia over the study period were 28.3 % (53/187) and 87.7 % (164/187), respectively. For infected calves, median time-to-onset for strongyles and coccidia was 78 (interquartile-range: IQR 38-117) and 43 (IQR 29-92) days, respectively. In the final Cox PH model for strongyles, breed (Ayrshires and Jerseys) and weaned calves had a greater hazard of infection than Friesians and preweaned calves, respectively. Calf tethering outside the pen was associated with a higher hazard of strongyle infection. In the final Cox PH model for coccidia, calves with watery and/or hemorrhagic diarrhea had a higher hazard compared with those with hard or soft feces. Weaning status and birth weight (kg) were time-varying covariates, leading to increased hazard over time. In the final MeNB model for strongyles, weaned animals had higher counts than those still on milk. In an interaction variable, the predicted strongyle-type egg counts increased with longer duration of farm operation when herd size was less than five cattle, but decreased when herd size was more than five. In the final MeNB model for coccidia, calves sometimes tethered outside their pens had higher counts than those continuously enclosed in the pen. Calf pen floors with either scant manure or moderate slurry had higher predicted counts than those on a clean pen floor. Calves with watery or hemorrhagic diarrhea and fed fresh Napier grass (Pennisetum purpureum) had higher counts compared with soft or hard feces and those not given fresh Napier, respectively. In an interaction variable, calves experiencing diarrhea and raised on elevated slatted floors had lower oocyst counts compared with those having diarrhea but not on elevated floors. The identified management practices associated with onset and counts of gastrointestinal parasitism should be considered in control efforts.

Effects of Calliandra and Sesbania on Daily Milk Production in Dairy Cows on Commercial Smallholder Farms in Kenya.

There is a growing interest in protein supplementation of dairy-cow diets using leguminous shrubs. The study objective was to ascertain the association between diet supplementation with Calliandra calothyrsus and Sesbania sesban and milk production in dairy cattle on commercial smallholder farms. This trial involved 235 cows from 80 smallholder dairy farms in Kenya randomly allocated to 4 intervention groups: (1) receiving Calliandra and Sesbania and nutritional advice; (2) receiving reproductive medicines and advice; (3) receiving both group 1 and 2 interventions; and (4) receiving neither intervention. Farm nutritional practices and management data were collected in a questionnaire, and subsequent physical examinations, mastitis tests, and milk production of cows on the farm were monitored approximately monthly for 16 months. Descriptive and univariable statistical analyses were conducted, and multivariable mixed-model regression was used for identification of factors associated (P < 0.05) with daily milk production. The mean milk production was 6.39 liters/cow/day (SD = 3.5). Feeding Calliandra/Sesbania to cows was associated (P < 0.0005) with an increase in milk produced by at least 1 liter/cow/day with each kg fed. Other variables positively associated with ln daily milk production in the final model included feeding of Napier grass, amount of silage and dairy meal fed, body condition score, and appetite of the cow. Other variables negatively associated with ln daily milk production in the final model included amount of maize germ fed, days in milk, sudden feed changes, pregnancy, and subclinical mastitis. In conclusion, our field trial data suggest that use of Calliandra/Sesbania through agroforestry can improve milk production in commercial smallholder dairy farms in Kenya. Agroforestry land use systems can be adopted as a way for dairy farmers to cope with feed shortages and low crude protein in farm-available feeds for their cows.

Effects of Calliandra and Sesbania supplementation on weight gain in dairy calves on smallholder farms in Kenya.

The growth rate of female calves on dairy farms is one of the crucial factors which influence age at first calving, affecting lifetime lactation productivity of a dairy cow. Diets with adequate crude protein are necessary to support calf growth. The study objective was to determine the effects of nutritional advice and diet supplementation with Calliandra calothyrsus and Sesbania sesban on average daily weight gain in dairy calves on semi-commercial smallholder dairy farms in Kenya. This trial involved 155 calves from 73 smallholder dairy farms, in Kenya, randomly selected and allocated to either intervention or control groups. The intervention group received nutritional advice and seedlings of Calliandra calothyrsus and Sesbania sesban, while the control group did not receive these interventions. Every 1-2 months for 16 months, data on farm nutritional practices and management were collected in a questionnaire, and physical examinations were done to monitor weight and health status. Descriptive and univariable statistical analyses were conducted, and multivariable mixed linear regression models were used for identification of factors associated (P < 0.05) with the natural log transformation of weight gain of calves on a given farm, controlling for clustering of visits within calves. Feeding at least 0.2 kg/day (wet weight) of Calliandra or Sesbania to calves <6 months old resulted in 33.2% increase in daily weight gain, while controlling for confounding by breed and sex of the calf. For calves ≥6 months, there was a significant interaction between amount of hay fed and if calves were also fed on Calliandra or Sesbania. When no Calliandra or Sesbania supplementation was provided, the mean weight gain was low and relatively constant even with increasing amounts of hay. When Calliandra or Sesbania supplement was added to the diet, the mean weight gain increased from 0.17 kg to 0.48 kg when hay was fed at 1 and 5 kg, respectively, while controlling for confounding by amount of maize silage fed and the prevailing season. In conclusion, supplementation of calf diets with at least 0.2 kg/calf/day of Calliandra or Sesbania is beneficial for faster weight gains.

Prevalence of lameness and associated risk factors on dairy farms in the Maritime Provinces of Canada.

Lameness in dairy cattle is a major issue for the industry due to the effects on the welfare of the animal, the economic impact, and consumer perception. The aim of this study was to determine the prevalence of lameness and explore potential risk factors in the Maritime Provinces of Canada. Cows were scored for lameness and potential risk factors and were assessed in 46 freestall herds and 33 tiestall herds in Nova Scotia, New Brunswick, and Prince Edward Island. In freestall herds, lameness was assessed using the most common method, locomotion scoring. A cow with a gait score of ≥3 out of 5 was considered to be lame. In tiestall herds, lameness was assessed using an alternative method known as stall lameness scoring. This assessment consisted of observation of the cow for 4 behavioral changes: standing on the edge of the stall, shifting weight, resting a limb, and uneven weight bearing when moved side to side. A cow displaying 2 or more of these behaviors was considered to be lame. At the time of the assessment, other animal-, environmental-, and management-based measurements were collected. These measurements were used in multivariable logistic regression analysis to determine risk factors that were associated with lameness for both freestalls and tiestalls independently. The prevalence of lameness was 21% for freestall-housed cattle and 15% for tiestall-housed cattle. Of the 1,488 tiestall-housed cows that were assessed, 68% showed no behavioral changes, whereas 15, 15, 2, and <1% showed 1, 2, 3, or 4 changes, respectively. In freestalls, higher odds of lameness were seen when cows spent ≥3 h/d in the holding area for milking compared with those that spent <3 h/d. In tiestall herds, higher odds of lameness were seen when bedding material was wet compared with when it was dry. For both lactating cow facility types, housing the dry cows and heifers on a deep bedded pack compared with tiestalls or freestalls was associated with a decreased odds of lameness. There were also many cow-level variables associated with lameness, including parity, daily milk production, stage of production, body condition, and width at the tuber coxae (hook bones). If producers become aware of the risk factors associated with lameness, they can make informed decisions on where to implement changes to help reduce the level of lameness in their herd.

Prevalence of hock, knee, and neck skin lesions and associated risk factors in dairy herds in the Maritime Provinces of Canada.

Skin lesions are commonly seen in dairy herds and have been associated with animal-, environmental-, and management factors. These lesions are not only a welfare concern, but they also affect profitability. Three areas on the cattle were examined for skin lesions: the hock, knee, and neck. Previous Canadian studies estimating the prevalence of lesions and the risk factors associated with them have not included the Maritime Provinces. In this study, 73 herds in the Maritime Provinces were chosen voluntarily to participate, with both tiestalls (n = 33) and freestalls (n = 40) represented. Within each herd, 67 to 90% of the lactating cows were selected and assessed for potential animal-, environmental-, and management-based risk factors. If producers were aware of the potential risk factors, this could help them reduce the prevalence in their herd. Leg lesions were scored on a 4-point scale (0-3) based on hair loss, swelling, and scabs, with a lesion defined as a score of 2 or 3 on at least 1 hock or knee. Necks were scored on a 3-point scale (0-2), with a lesion defined as score 2. For freestalls, the prevalence (95% confidence interval) of hock lesions was 39% (29-49%), knee lesions was 14% (11-18%), and neck lesions was 1% (<1-2%). Similarly, for tiestalls the prevalence (95% confidence interval) of hock lesions was 39% (33-46%), knee lesions was 17% (13-22%), and neck lesions was 5% (3-8%). Due to differences in management and methods of assessment between facility types, tiestalls and freestalls were analyzed separately. Due to dichotomization of cows as having a skin lesion or not, random-effects multivariable logistic regression was used to determine the risk factors for each lesion and facility type. Several environmental-based measurements, such as the stall base, type and dryness of bedding, and type of milking parlor, were associated with leg lesions. An environmental-based measurement that was associated with neck lesions was the design of the feed rail barrier in freestalls and the dimensions of the tie rail in tiestalls. Animal-based risk factors, such as stage of lactation, parity, and body condition, were also associated with all 3 types of lesions. This study showed that lesions to the hock, knee, and neck were common in the Maritime Provinces of Canada. Although differences were seen between facility types, in general, the results suggest that improving stall design and management and feed bunk design would help producers reduce the number of skin lesions seen in dairy cattle.

Animal and management factors associated with weight gain in dairy calves and heifers on smallholder dairy farms in Kenya.

Calf growth is an important determinant of dairy herd productivity, particularly in countries where the dairy industry is expanding, such as Kenya. Our objective was to determine factors associated with weight gain in randomly selected dairy calves and heifers in smallholder dairy farms (SDF) in Kenya. A cross-sectional study on a census of 321 calves and heifers (dairy calves and heifers up to 36 months of age), sampled from 200 randomly selected SDF in Naari, Kenya, formed the study population. Youngstock management was recorded using a questionnaire. Biodata were obtained through subsequent physical examination and heart girth measurement. Descriptive statistical analyses were conducted, and mixed model regression was used for identification of factors associated (p < 0.05) with the natural log transformation of estimated average daily weight gain (ADG). Median and mean ADG of the youngstock were 360 and 443 (s.d. = 375) g day-1, respectively. In the final model, ADG was highest in pre-weaned calves and declined with age. Supplementing with quality hay during the dry season at least weekly was associated with increased ADG. There was an interaction between breed and historical disease on ADG such that disease was associated with decreased ADG in Bos taurus breeds, while ADG in Bos indicus breeds was not affected by disease. There was a significant interaction between education levels of the husband and wife caretakers; when the man's education was low (having less than or equal to primary school), ADG was highest when the woman had not completed primary school, but was lower when the woman had completed primary, secondary or tertiary education, possibly because such women worked off-farm more often. General growth performance of animals on these farms was lower than benchmarked standards recommended for optimum dairy production but within previously reported ranges for the East African region. Supplementation of diets (with hay and/or concentrates) is recommended for optimum growth in calves and heifers, especially in the dry season. If nutritional management of Bos taurus youngstock is not improved, crosses of Bos indicus could be better suited for the current nutritional management systems in SDF in Meru having calf disease problems. Interventions to support educating women (the primary caretakers of the cattle) and men in the community on calf management should be initiated, preferably with shared on-farm responsibilities. Training in better management, even for learned farmers, would be critical to better calf growth.

Comparison of fecal pooling strategies for detection of Mycobacterium avium ssp. paratuberculosis in cattle.

In herds with typical moderate to low within-herd prevalence, testing for Mycobacterium avium ssp. paratuberculosis (MAP), the infectious agent of Johne's disease, will be more cost-effective if individual fecal samples are cultured in composite pools. However, sensitivity to classify a pool containing 1 or more positive individual samples as positive may depend on pool size and number of individual positive samples within a pool. Fecal samples collected from 994 dairy cows sampled at slaughter were cultured to detect MAP. Culturing was done both individually and as composite pooled samples using the TREK ESP Culture System II broth medium (Thermo Fisher Scientific, Trek Diagnostic Systems Inc., Cleveland, OH). Composite samples consisted of pools containing feces from 3, 5, 8, 10, or 15 cows. The number of individual fecal culture-positive cows within each pool ranged from 0 to 4. Culture of individual fecal samples detected MAP in 36 (3.6%) of the 994 cows. Individual samples that were detected within the first 50 d by TREK ESP Culture System II were more likely to lead to a positive pool result. In total, 840 pooled fecal samples were examined for presence of MAP, and of those, 272 pools actually contained feces from fecal culture-positive cows. The crude sensitivity (proportion of pools that contained at least 1 fecal-positive cow that tested positive) for pools of 3, 5, 8, 10, and 15 was 47, 67, 44, 59, and 39%, respectively. Across pools, an increase of the number of fecal culture-positive samples from 1 to 2 enhanced overall crude sensitivity from 44 to 71%. However, sensitivity did not further increase for pools with 3 or 4 fecal culture-positive samples (63 and 60%, respectively). Additionally, a simulation analysis assessing probability of pooled fecal samples being positive in herds of 50 and 100 cows was conducted. The simulation assumed that 1, 2, or 5 cows per herd were MAP fecal culture-positive and that pools of 5 and 10 were used. This low-prevalence herd simulation indicated that weighted mean herd probabilities of detecting a positive herd ranged between 52 and 99.3%, with the lowest probability for pools of 10 with 1 positive cow in the herd and the highest probability for pools of 5 with 5 positive cows in the herd. However, overall, pools of 5 and 10 had similar diagnostic capabilities, enabling cost savings by utilizing pools of 10.

Pulmonary outcomes following specialized respiratory management for acute cervical spinal cord injury: a retrospective analysis.

STUDY DESIGN: Retrospective analysis. OBJECTIVES: To identify multivariate interactions of respiratory function that are sensitive to spinal cord injury level and pharmacological treatment to promote strategies that increase successful liberation from mechanical ventilation. SETTING: United States regional spinal cord injury (SCI) treatment center. METHODS: Retrospective chart review of patients consecutively admitted to Santa Clara Valley Medical Center between May 2013 and December 2014 for ventilator weaning with C1-C5 American Spinal Injury Association Impairment Scale (AIS) A or B SCI, <3 months from injury and who had a tracheostomy in place. A nonlinear, categorical principal component analysis (NL-PCA) was performed to test the multivariate interaction of respiratory outcomes from patients (N=36) being weaned off ventilator support after acute SCI with (N=15) or without (N=21) theophylline treatment. RESULTS: In total, 36 patients met inclusion criteria (2 C1, 5 C2, 11 C3, 14 C4 and 4 C5). The NL-PCA returned three independent components that accounted for 95% of the variance in the data set. Multivariate general linear models hypothesis tests revealed a significant syndromic interaction between theophylline treatment and SCI level (Wilks' Lambda, P=0.028, F (12,64)=2.116, η2=0.256, 1-ß=0.838), with post hoc testing demonstrating a significant interaction on PC1, explained by a positive correlation between improved forced vital capacity and time it took to reach 16 h of ventilator-free breathing. Thirty-three patients (92%) achieved 16 h of ventilator-free breathing (VFB) and 30 patients (83%) achieved 24 h of VFB. CONCLUSIONS: We suspect that some portion of the high success rate of ventilator weaning may be attributable to theophylline use in higher cervical SCI, in addition to our aggressive regimen of high volume ventilation, medication optimization and pulmonary toilet (positive pressure treatments and mechanical insufflation-exsufflation).

Evaluation of selective dry cow treatment following on-farm culture: Milk yield and somatic cell count in the subsequent lactation.

Compared with blanket dry cow therapy (DCT), the selective antimicrobial treatment of cows based upon on-farm culture results has the potential to reduce the amount of antimicrobials used in dairy production. The objective of the current study was to determine the effect of a Petrifilm (3M Canada, London, Ontario) on-farm culture-based selective DCT program on milk yield and somatic cell count (SCC) in the following lactation. A total of 729 low-SCC (<200,000 cells/mL) cows from 16 commercial dairy herds with a low bulk tank SCC (<250,000 cells/mL) were randomly assigned to receive either blanket DCT or Petrifilm-based selective DCT. Cows belonging to the blanket DCT group were infused with a commercial DCT product and an internal teat sealant (ITS) at drying off. Using composite milk samples collected on the day before drying off, cows in the selective DCT group were treated at drying off based on the results obtained by the Petrifilm on-farm culture system with DCT and ITS (Petrifilm culture positive) or ITS alone (Petrifilm culture negative). Milk test-day records for the following lactation were obtained from Dairy Herd Improvement for all cows enrolled in the trial. Repeated measures linear mixed models were used to assess the effect of study group (blanket or selective DCT) on test-day milk production and natural logarithm of SCC over the first 180 d of the subsequent lactation. According to the final multivariable models, when low-SCC cows were selectively treated with DCT at drying off based on results obtained using the Petrifilm on-farm culture system, no effect on milk production (least squares means for blanket DCT = 39.3 kg vs. selective DCT = 39.0 kg) or natural logarithm of SCC (least squares means for blanket DCT = 3.95 vs. selective DCT = 3.97) was observed in the subsequent lactation when compared with cows receiving blanket DCT. The results of this study indicate that selective DCT based on results obtained by the Petrifilm on-farm culture system enabled a reduction in the use of DCT without negatively affecting milk production and milk quality.

Evaluation of selective dry cow treatment following on-farm culture: risk of postcalving intramammary infection and clinical mastitis in the subsequent lactation.

The objective of the study was to evaluate the utility of a Petrifilm-based on-farm culture system when used to make selective antimicrobial treatment decisions on low somatic cell count cows (<200,000 cells/mL) at drying off. A total of 729 cows from 16 commercial dairy herds with a low bulk tank somatic cell count (<250,000 cells/mL) were randomly assigned to receive either blanket dry cow therapy (DCT) or Petrifilm-based selective DCT. Cows belonging to the blanket DCT group were infused with a commercial dry cow antimicrobial product and an internal teat sealant (ITS) at drying off. Using composite milk samples collected on the day before drying off, cows in the selective DCT group were treated at drying off based on the results obtained by the Petrifilm on-farm culture system with DCT + ITS (Petrifilm culture positive), or ITS alone (Petrifilm culture negative). Quarters of all cows were sampled for standard laboratory bacteriology on the day before drying off, at 3 to 4d in milk (DIM), at 5 to 18 DIM, and from the first case of clinical mastitis occurring within 120 DIM. Multilevel logistic regression was used to assess the effect of study group (blanket or selective DCT) and resulting dry cow treatment (DCT + ITS, or ITS alone) on the risk of intramammary infection (IMI) at calving and the risk of a first case of clinical mastitis between calving and 120 DIM. According to univariable analysis, no difference was observed between study groups with respect to quarter-level cure risk and new IMI risk over the dry period. Likewise, the risk of IMI at calving and the risk of clinical mastitis in the first 120 DIM was not different between quarters belonging to cows in the blanket DCT group and quarters belonging to cows in the selective DCT group. The results of this study indicate that selective DCT based on results obtained by the Petrifilm on-farm culture system achieved the same level of success with respect to treatment and prevention of IMI over the dry period as blanket DCT and did not affect the risk of clinical mastitis in the first 120 d of the subsequent lactation.

Evaluation of milk ELISA for detection of Mycobacterium avium subspecies paratuberculosis in dairy herds and association with within-herd prevalence.

Cow-level milk ELISA results can be used to determine herd Mycobacterium avium ssp. paratuberculosis (MAP) status. Milk sample collection is minimally invasive and ELISA results can be obtained quickly and economically. The objectives were to evaluate the herd-level test characteristics of 3 commercial milk ELISA, and to determine the impact of within-herd MAP prevalence on the performance of the milk ELISA herd test. A total of 32 purposively selected herds with a median herd size of 66 milking cows were used in this 2-yr project. Fecal and milk samples were collected from all milking cows at 6-mo intervals. Fecal samples were pooled by cow age, with 5 cow samples per pool; individual fecal culture was completed on cow samples from positive pools. Herd MAP status was defined as MAP positive if, at any point during the longitudinal study, a pooled fecal culture from the herd was positive. Milk samples were analyzed using each of 3 commercial milk ELISA kits; a cow-level result from each ELISA was classified as positive following the respective manufacturer's recommended threshold for a positive result. Herd-level milk ELISA test characteristics were estimated using generalized estimating equations logistic models, which accounted for repeated measurements. Using a cutoff of 2% milk ELISA-positive cows, milk ELISA herd sensitivity relative to a herd MAP status based on all pooled fecal culture results collected during the study was as follows: ELISA A: 59% [95% confidence interval (CI): 36-78%), ELISA B: 56% (95% CI: 32-77%), and ELISA C: 63% (95% CI: 41-81%). Herd specificity for ELISA A, B, and C was 80% (95% CI: 71-88%), 96% (95% CI: 89-98%), and 92% (95% CI: 86-96%), respectively. The remainder of the analyses focused on results from ELISA B. Herd sensitivity of ELISA B increased as MAP prevalence increased. In herds with a mean MAP prevalence ≤5%, the herd sensitivity of the milk ELISA was low, ranging from 11% when MAP prevalence was 1%, to 62% when MAP prevalence was 5%. Categorical likelihood ratios based on milk ELISA within-herd prevalence predicted that herds with milk ELISA prevalence above 0 but <2% had a similar likelihood to be MAP positive or MAP negative, whereas herds with a milk ELISA prevalence between 2 and 4% were 3.7 times more likely to be MAP positive than MAP negative. All herds with a milk ELISA prevalence >4% were MAP positive. Although milk ELISA B worked well to establish herd MAP status in high-prevalence herds, interpretation was unreliable in MAP-negative and low-prevalence herds.

Evaluation of a 3M Petrifilm on-farm culture system for the detection of intramammary infection at the end of lactation.

The purpose of this study was to evaluate a 3M Petrifilm-based on-farm culture system for the detection of intramammary infection (IMI) in low somatic cell count (SCC) cows (<200,000 cells/mL) at drying off. The main objectives were to determine the test characteristics and the predictive values of the Petrifilm on-farm culture system. The ability of dairy producers to correctly classify cows as infected or uninfected based on Petrifilm culture and a set colony count threshold was also assessed. A total of 360 cows originating from 16 low bulk tank SCC (<250,000 cells/mL) dairy herds were enrolled at drying off. Enrolled cows had an expected dry period of 30-90 days, a SCC<200,000 cells/mL on the last 3 tests prior to drying off, no clinical mastitis in the same time period, and no antibiotic treatment in the last 14 days. Quarter milk samples were collected on the day prior to drying off, and a composite milk sample was created by combining 5 mL of milk from each quarter sample. Composite milk samples were cultured on-farm using the Petrifilm culture system, which provided results within 24h. Quarter milk samples were cultured in a reference laboratory, and the results were aggregated to the cow level. On the day of drying off, the Petrifilm was read by the producer and cows were classified as positive if ≥5 colonies (equivalent to 50 colony forming units/mL) were present. When read by the producer, 47.8% of the cows cultured negative on Petrifilm and were infused with only an internal teat sealant at drying off. The test characteristics of the Petrifilm on-farm culture system were calculated by comparing the producer-derived Petrifilm results to those obtained by standard laboratory culture. The sensitivity and specificity of the Petrifilm on-farm culture system were 85.2% (78.5-90.5) and 73.2% (66.4-79.3), respectively. The negative predictive value of the Petrifilm test system was high (86.6%) when estimated using the prevalence of IMI in this data set, and the positive predictive value was moderate (70.9%). An automated 3M Petrifilm reader was used to obtain accurate colony counts. The agreement between Petrifilm results obtained by the producer and those obtained by the automated Petrifilm reader was high, with a kappa value of 0.82 (0.75-0.89).

The BH3 mimetic HA14-1 enhances 5-fluorouracil-induced autophagy and type II cell death in oesophageal cancer cells.

BACKGROUND: Resistance to chemotherapeutic agents has been associated with a failure of cancer cells to induce apoptosis. Strategies to restore apoptosis have led to the development of BH3 mimetics, which inhibit anti-apoptotic Bcl-2 family members. We examined the sensitivity of three oesophageal cancer cell lines to 5-fluorouracil (5-FU) alone and in combination with the BH3 mimetic HA14-1. METHODS: Clonogenic assays, morphology, markers of autophagy and apoptosis were used to assess the involved death mechanisms. RESULTS: In response to 5-FU treatment, OE21 cells induce apoptosis, KYSE450 and KYSE70 cells are more resistant and induce autophagy accompanied by type II cell death. Autophagy induction results in ineffective treatment as substantial numbers of cells survive and re-populate. HA14-1 did not improve 5-FU treatment or reduce colony re-growth in the apoptosis deficient KYSE70 cells. However, the sensitivity of OE21 (apoptotic) and KYSE450 cells (apoptosis deficient/type II cell death) was significantly improved. In OE21 cells, treatment with 5-FU and HA14-1 resulted in augmentation of apoptosis. In KYSE450 cells, the reduction in recovering colonies following combination treatment was due to the enhancement of type II cell death. CONCLUSION: The efficacy of HA14-1 is cell line dependent and is not reliant on apoptosis induction.

Curcumin induces apoptosis-independent death in oesophageal cancer cells.

BACKGROUND: Oesophageal cancer incidence is increasing and survival rates remain extremely poor. Natural agents with potential for chemoprevention include the phytochemical curcumin (diferuloylmethane). We have examined the effects of curcumin on a panel of oesophageal cancer cell lines. METHODS: MTT (3-(4,5-dimethyldiazol-2-yl)-2,5 diphenyl tetrazolium bromide) assays and propidium iodide staining were used to assess viability and DNA content, respectively. Mitotic catastrophe (MC), apoptosis and autophagy were defined by both morphological criteria and markers such as MPM-2, caspase 3 cleavage and monodansylcadaverine (MDC) staining. Cyclin B and poly-ubiquitinated proteins were assessed by western blotting. RESULTS: Curcumin treatment reduces viability of all cell lines within 24 h of treatment in a 5-50 muM range. Cytotoxicity is associated with accumulation in G2/M cell-cycle phases and distinct chromatin morphology, consistent with MC. Caspase-3 activation was detected in two out of four cell lines, but was a minor event. The addition of a caspase inhibitor zVAD had a marginal or no effect on cell viability, indicating predominance of a non-apoptotic form of cell death. In two cell lines, features of both MC and autophagy were apparent. Curcumin-responsive cells were found to accumulate poly-ubiquitinated proteins and cyclin B, consistent with a disturbance of the ubiquitin-proteasome system. This effect on a key cell-cycle checkpoint regulator may be responsible for the mitotic disturbances and consequent cytotoxicity of this drug. CONCLUSION: Curcumin can induce cell death by a mechanism that is not reliant on apoptosis induction, and thus represents a promising anticancer agent for prevention and treatment of oesophageal cancer.

Evaluation of the University of Minnesota Tri-plate and 3M Petrifilm for the isolation of Staphylococcus aureus and Streptococcus species from clinically mastitic milk samples.

The primary objective was to compare microbiological results of the University of Minnesota Tri-plate and the 3M Petrifilm Staph Express (STX) Count Plate to standard culture techniques for identification of clinical mastitis caused by Staphylococcus aureus. The secondary objective was to evaluate the Tri-plate's ability to differentiate Streptococcus spp. from other gram-positive organisms. The tests were evaluated using clinically positive mastitic milk samples (n = 282) to determine their ability to diagnose the pathogens of interest. A Tri-plate was classified positive for Staph. aureus when at least 1 colony exhibiting beta-hemolysis was present on the Factor media portion of the plate. When the plate was used in this manner and read by a trained laboratory technician, the sensitivity of the Tri-plate was 97.9% and the specificity was 81.8%. When the Tri-plate was evaluated by the laboratory technician for its ability to diagnose Streptococcus spp., both sensitivity and specificity of the test were very good (92.6 and 89.5%, respectively). Using the Petrifilm, samples were classified as positive for Staph. aureus if any red-violet colonies were present on the Petrifilm after an initial 24-h incubation. When used in this manner, the Petrifilm had a sensitivity of 97.4% and a specificity of 76.1%. Further evaluation of the Petrifilm was done using the STX disk, which was used to confirm the presence of Staph. aureus. When using the presence of 1 pink colony on the disk, the sensitivity of the Petrifilm was 92.1% and the specificity was 93.1%. Both the Tri-plate and the 3M STX Petrifilm successfully diagnosed Staph. aureus in clinical milk samples when used in a laboratory setting and the Tri-plate successfully differentiated Streptococcus spp. from other gram-positive organisms.

Laboratory evaluation of 3M Petrifilms and University of Minnesota Bi-plates as potential on-farm tests for clinical mastitis.

The objective was to determine test characteristics and compare 2 potential on-farm culture systems for clinical mastitis, the Minnesota Easy Culture System II Bi-plate and Petrifilm. The tests were evaluated using clinically positive mastitic milk samples (n = 282) to determine their ability to differentiate appropriate treatment groups; all cases that had gram-positive growth were considered treatment candidates (n = 161), whereas cases that grew gram-negative organisms only or yielded no bacterial growth were classified as no treatment (n = 121). For Petrifilm, both undiluted and 1:10 diluted milk samples were used. To create treatment categories, 2 types of Petrifilms were used, Aerobic Count (AC) and Coliform Count (CC). Both Bi-plates and Petrifilms were read after 24 h of incubation. Analysis was conducted at various colony count thresholds for the Petrifilm test system. The combination of Petrifilms that had the highest sensitivity classified a case as gram-negative if there were > or =20 colonies present on the CC. If there were <20 colonies present on the CC and >5 colonies present on the AC, a case would be classified as gram-positive. The Bi-plate had a sensitivity of 97.9% and a specificity of 68.6%. The Petrifilm test system had a sensitivity of 93.8% and a specificity of 70.1%. There was no significant difference in the sensitivities between the tests. All Bi-plates and Petrifilms were read by a laboratory technician and a group of masked readers with limited microbiology training. Kappa values for the masked readers were 0.75 for Bi-plates and 0.84 and 0.86 for AC and CC Petrifilms, respectively. The Bi-plate and Petrifilm were able to successfully categorize clinical cases of mastitis into 2 treatments based on their ability to detect the presence of a gram-positive organism. Neither method had the ability to determine if a sample was contaminated. The results of this study indicate that both tests were able to appropriately categorize cases, which could potentially result in a reduction in the quantity of antibiotics used to treat clinical cases of mastitis.

Cyclooxygenase-2 inhibitors demonstrate anti-proliferative effects in oesophageal cancer cells by prostaglandin E(2)-independent mechanisms.

The incidence of oesophageal cancer (OC) has risen in recent decades, with survival rates remaining poor despite surgical treatment and adjuvant chemotherapy. Studies have reported cyclooxygenase-2 (COX-2) overexpression in OC and current evidence suggests NSAIDs have major potential for chemoprevention through COX-2 inhibition. However, several reports have questioned the specificity of these inhibitors, suggesting they may act through mechanisms other than COX-2. We evaluated the effects of specific COX-2 inhibitors, NS-398 and nimesulide, on cell lines of both histological types of OC. COX-2 protein expression varied in the cell lines and corresponded with levels of prostaglandin E(2) (PGE(2)) production. Following treatment with low concentrations of NS-398 (0.1 microM), PGE(2) production was reduced dramatically, indicating inhibition of COX-2 activity. Examination of cellular morphology, caspase-3 activity and mitochondrial membrane integrity found no major induction of apoptotic cell death at concentrations below 100 microM. Tumour cell proliferation was significantly reduced at high concentrations (50-100 microM) of both inhibitors over 6 days. Cellular responses were more evident in NS-398-treated adenocarcinoma cells. However, concentrations required to inhibit proliferation were up to 1000-fold higher than those needed to inhibit enzyme activity. Addition of exogenous PGE(2) to NS-398-treated adenocarcinoma cells failed to reverse the inhibitory effects, indicating PG and COX-2 independence. It remains possible that in vivo COX-2 is the primary target, as enzyme inhibition can be achieved at low concentrations, however, inhibition of proliferation is not the primary mechanism of their anti-tumour activity.